Congenital Myasthenic Syndromes (CMS) are a group of inherited disorders resulting from abnormal signal transmission of NMJ at different levels of the presynaptic, synaptic and postsynaptic apparatus. Up to now, more than 30 genes have been found to underlie CMS and the differentiation of CMS subtypes, usually clinically heterogenous, can be performed by molecular genetic tests (29, 30). DOK7-CMS is reported to occur in up to almost 20% of CMSs (19). The DOK7 protein, which is encoding by DOK7 gene, is involved in the normal development and maintenance of the NMJ (18, 31). DOK7-associated CMS mutations are widely accepted as being inherited in an autosomal recessive pattern. However, this is not always the cases (32). A variety of mutations in the DOK7 gene are reported to be the cause of CMS with the most common (more than 65%) mutation being c.1124_1127dupTGCC in exon 7, which is in homozygous or compound heterozygous form (16, 33). This variant results in a truncated protein and leads to the loss of two tyrosine residues that are phosphorylated and recruits CRK proteins, which are important for anchoring acetylcholine receptors at synapses.
In this study, in addition to reporting a novel disease-causing variant in DOK7 gene, we describe a late-onset form of CMS in 5 carriers of this variant. Whereas a late-onset presentation of CMS is uncommon, it does not rule out CMS (Supplementary Table 2&3). Due to the overlap of clinical features, during the initial diagnosis process, it may be misdiagnosed as limb-girdle weakness or myasthenia gravis (34). Thus, late-onset CMS is probably still underdiagnosed in plenty of cases (35). Despite the fact that our homozygous patients have the same genotype, their manifestations of the disease were not the same. This may be due to their different genetic background. Congenital bilateral ptosis and facial and muscle weakness were observed in the primary proband, while in the cases VI-1 and VI-2, unilateral ptosis in different eyes (Fig. 5C) and muscle weakness onset were noticeable during childhood (at the age of about 6 years). However, all of them had VCP. This feature is reported in CMS patients with mutations in COLQ and DOK7 genes, as well as patients with MUSK deficiency (36–38). Therefore, genetic testing is essential to identify the etiology and could have a role in the proper management of the disease. Furthermore, the severity of muscle weakness in all homozygous patients was mostly variable. Thus, there was no consistent genotype-phenotype correlations. Moreover, the clinical features in the heterozygous cases with late-onset manifestations were somewhat different. The facial weakness, mild ptosis and lack of strength in arms and shoulders with different severity after the age of 40 were mostly common, while, the respiratory failure was only seen in the case IV-4 after the age of 50 years (none of other carriers have reached this age yet). Meanwhile, there are reports of several cases of DOK7-CMS with neonatal or childhood respiratory failure (4, 18, 33), none of our homozygous patients showed this feature. However, it was the first report of the late-onset CMS in patients with a heterozygous pathogenic variant in DOK7 gene in Iran, there are a few reports of late-onset presentations with clinical heterogeneity in carriers, but the precise mechanism is unexplained (2, 39). The phenotypic presentations and intra-and inter-familial variable expressivity of CMS in carries of this study may be related to the dominant-negative effects of the identified variant or haploinsufficiency mechanism as a consequence of partial autosomal dominant inheritance. A late-onset CMS patient caused by heterozygous mutation in the DOK7 gene, which was previously described by Bastos et al, represented the symptoms of the disease after the age of 65 and the first features were increased difficulty in climbing stairs, weakness on both legs, lack of strength in both arms and finally sudden respiratory failure without ptosis and facial palsy (2). Thus, the clinical presentations of late-onset CMS would not be similar, even if the mutated gene is the same.
Simulation analysis of this study showed that mutation in the DOK-W induces the variations in the structures and molecular dynamic parameters and subsequently instability and dysfunction of the protein. The mean of RMSD indicated that simulation system is stabled in the 200 ns of simulation time. However, decrease in RMSD for DOK-M compared to DOK-W demonstrated that this mutation affects the protein structure (40). Also, a comparison of Rg data of both proteins revealed the more compactness in DOK-M which might lead to decreased stability and functional impairment of the DOK-M (41). The different variations in the RMSF parameter of amino acid residues in the DOK-M in comparison of DOK-W shows the impact of this mutation on amino acids’ fluctuation. Given that increment of TE in the mutant form of the protein increases the intermolecular energy, molecular instability could ensue. Moreover, changes in the DOK-M structure decrease the mean of H-bonds compared with DOK-W. Decrease in the intermolecular hydrogen bonds can cause the protein stability reduction (42). Finally, the variation of secondary structure of the protein and induction of changes in the mean of α-Helix, β-Sheet, Coil, Bend, and Turn parameters between the DOK-M and DOK-W during the simulation time showed that this mutation induced remarkable conformational changes in the wild type of DOK protein, subsequently influencing the folding and function of the protein (43).
A variety of therapeutic interventions are available, which are beneficial in treating some kinds of CMSs, but accurate genotype diagnosis is important for the disease management (28, 31, 44). Ephedrine (a sympathomimetic with α- and β-adrenergic effects) and salbutamol (a selective β2 agonist) were very effective therapeutic agents in some CMS patients, especially those with COLQ, laminin-β2, DOK7, MUSK, Agrin and plectin-1 deficiency, and provided more benefit in types of CMS, which are not effectively treated by acetylcholinesterase inhibitors (2, 45–48). β2-Adrenergic agonists, partially compensate disrupted postsynaptic structures. Therefore, a remarkable response is seen in CMS subtypes with mutations in genes encoding proteins that are involved in formation and stability of the NMJ within the LRP4-MUSK-DOK7 signaling pathway (49, 50). In an in vivo study on DOK7-CMS model mice, salbutamol treatment increased the number of active NMJs as well as increasing muscle strength and prolonged the survival of these myasthenic mice (51). The precise selection of drug therapy is very important, as the same drug can be effective, ineffective, or even worsen the clinical features in different types of CMS (52)(Supplementary Table 1). In the present study, when the clinical symptoms of the disease were obvious, these patients experienced a short period of ephedrine. This treatment not only did not improve their symptoms but also worsened the condition of all homozygous patients in a short time. Therefore, ephedrine treatment was discontinued after 7 days. After that, when the pathogenic variant in the DOK7 gene was identified by ES, the treatment with salbutamol was started, and the dramatical improvement of the patients' condition was observed in a very short period of time. The peak of treatment effect with ephedrine and salbutamol was reported to be about 6–8 months after start using these drugs (53), in contrast to anticholinesterases, full effects of which were not immediate (25). Only after 3 days of using salbutamol, our homozygous patients started doing their daily tasks. Walking and climbing up the stairs got better and their ptosis gradually improved. Eventually during our 18 months follow-up a significant improvement was observed. While headache, tremor and occasional palpitations were reported as side effects of salbutamol in some cases (24), no side effects were observed in our patients after using it. We also started salbutamol therapy (in a different dose) in 5 carriers, who were showing late-onset form of CMS (supplementary table 1), and its very beneficial effects were obtained. In cases IV-4, V-2, V-8, V-10 and V-11, there was a significant increase in the strength of the muscles, a decrease in the facial weakness, and a prominent improvement in their ptosis in an 18-month follow-up. Also, no recurrence of respiratory failure was observed in case IV-4, after receiving salbutamol. It has been proven that in the treatment with salbutamol, the age of the disease onset, the age at treatment start and the drug dosage have no effect on the treatment outcome (53). Our literature review showed that in addition to the aforementioned factors, gender is also ineffective in responding to treatment with this drug, and both sexes, regardless of the age of onset of the disease, responded well to salbutamol therapy (48, 54–56)(Fig. 6) (Supplementary Table 2&3). Interestingly, although the mechanism of action of ephedrine and salbutamol are probably similar, in our patients, ephedrine had a negative effect on the disease, while salbutamol had completely positive consequences. There are some reports of other studies which the ephedrine therapy in DOK7-CMS was not efficient, but herein we are reporting negative effects of ephedrine in our patients (Supplementary Table 1).